Combination of Highly Efficient Hexapeptide Ligand Library-Based Sample Preparation with 2D DIGE for the Analysis of the Hidden Human Serum/Plasma Proteome - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 172

2.3. Protein Labeling
(Minimal Labeling
with CyDyes)

1. CyDyes: Cy2, Cy3, and Cy5; DIGE minimal labeling kit (GE
Healthcare, Freiburg, Germany). These dyes are shipped
lyophilized and should be stored under light protection at
−20°C.
2. Anhydrous dimethylformamide (DMF; see Note 5).
3. 10 mM Lysine solution.

1. IPG strips (linear pH 4-7, GE Healthcare).
2. IPG buffer corresponding to the used pH range (pH 4-7, GE
Healthcare).
3. Rehydration buffer: 2% (w/v) CHAPS, 0.4% (w/v) DTT, 7 M
urea, 2 M thiourea, and 0.5% (v/v) corresponding IPG buffer
(see Note 4).
4. 2D DIGE lysis buffer: 25 mM Tris, 4% (w/v) CHAPS, 7 M
urea, and 2 M thiourea.
5. Equilibration buffer with DTT: 50 mM Tris-HCl (pH 8.8),
6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.5% (w/v)
DTT (see Note 6).
6. Equilibration buffer with iodoacetamide: 50 mM Tris-HCl
(pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and
4.5% (w/v) iodoacetamide (see Note 6).
7. Polyacrylamide gels with corresponding buffer system (in our
case large-format 12.5%-SDS-PAGE gels with 50 mM
Tris/192 mM glycine buffer system; see Note 7).

2.4. Isoelectric
Focusing and Large-
Format SDS PAGE

1. Laser scanner with 488 nm-, 532 nm- and 633 nm-excitation
wavelength and a resolution of at least 100

2.5. Imaging
and Image Analysis m is essential. If

2D DIGE gels are scanned between the glass plates, a confocal
optic is recommended for the scanner (specifi cations are ful-
fi lled by the Typhoon 9400; GE Healthcare). Alternatively,
CCD-based camera systems with suffi cient resolution and mul-
tiplexing capabilities can be used.
2. Lint-free paper (see Note 8).
3. 2DE image analysis software package compatible with DIGE
multi-channel images and the corresponding scanner fi le for-
mat (e.g., DeCyder, Proteomweaver).

μ

3. Methods

Here we describe a protocol for reproducible and easy-to-use
“one-step” sample preparation of blood samples utilizing hexapep-
tide ligand libraries. Due to its proven applicability for quantitative
proteomic approaches, it is obvious to combine it as front-end
sample preparation for DIGE profi ling, which is widely accepted